We propose to investigate the functional significance of DNA-damage-induced, ATM/ATR-dependent phosphoryIation of the major cellular single-stranded DNA-binding protein, RPA. Since RPA is essential for DNA replication and DNA repair, phosphorylation-dependent alteration in RPA function has the potential of altering the cellular responses to DNA damage. Indeed, we have demonstrated that DNA damage-induced ATM-dependent phosphorylation of RPA occurs, and it changes RPA function in vitro. Specifically, RPA phosphorylation alters DNA binding activity and protein/protein interactions. We postulate that a failure to phosphorylate RPA and other proteins required for DNA replication and DNA repair in ATM mutants compromises the cellular switch from DNA replication to DNA repair that would normally protect cells from the deleterious consequences of DNA damage. We propose that these abnormal responses in A-T lead to the enhanced cell death and genetic instability associated with the disease. We propose to test the hypothesis that the failure to phosphorvlate RPA (and other DNA repair proteins) in response to DNA damage compromises the DNA repair capacity of A-T ceils. The specific aims of this project are: 1. To determine the sites of ATM- and ATR-mediated RPA phosphorylation and construct site-specific mutants to test their role in RPA function. 2. To determine the influence of RPA (and mutant RPA) phosphorylation on protein/protein interactions in vivo and in vitro. A newly-developed RPA phospho-specific antibody will greatly facilitate these studies. 3. To clarify the role of the ATM kinase activity vs. other ATM functions in DSB repair: We are concentrating on abnormal responses to DNA damage in A-T cells that may influence the DNA repair capacity of these cells and may contribute to the high cancer risk and neurodegeneration. The ultimate goal of this project is to improve our understanding of the mechanisms that cells use to maintain genomic stability. This improved understanding may ultimately lead to the development of strategies for preventing and/or treating diseases that are characterized by genomic instability and neurological defects.